Tumor Biology

, Volume 36, Issue 4, pp 3025–3033 | Cite as

D-loop somatic mutations and ∼5 kb “common” deletion in mitochondrial DNA: important molecular markers to distinguish oral precancer and cancer

  • Sayantan Datta
  • Esita Chattopadhyay
  • Jay Gopal Ray
  • Mousumi Majumder
  • Puspita Das Roy
  • Bidyut Roy
Research Article

Abstract

Apart from genomic DNA, mutations at mitochondrial DNA (mtDNA) have been hypothesized to play vital roles in cancer development. In this study, ∼5 kb deletion and D-loop mutations in mtDNA and alteration in mtDNA content were investigated in buccal smears from 104 healthy controls and 74 leukoplakia and 117 cancer tissue samples using Taqman-based quantitative assay and re-sequencing. The ∼5 kb deletion in mtDNA was significantly less (9.8 and 10.5 folds, P < 0.0001) in cancer tissues compared to control and leukoplakia tissues, respectively. On the other hand, somatic mutations in D-loop, investigated in 54 controls, 50 leukoplakias and 56 cancer patients, were found to be significantly more in cancer tissues, but not in leukoplakia tissues, compared to control (Z-score = 5.4). MtDNA contents were observed to be significantly more in leukoplakia (2.1 folds, P = 0.004) and cancer (1.6 folds, P = 0.03) tissues compared to control tissues. So, D-loop somatic mutations and ∼5 kb deletion patterns could be used as distinguishing markers between precancer and cancer tissues. This observation further suggests that somatic mutations in D-loop may facilitate carcinogenesis and cancer cells with less ∼5 kb deletion, i.e., intact mtDNA, may become resistant to apoptosis.

Keywords

Oral precancer Cancer ∼5 kb deletion D-loop mutation 

Abbreviations

HVS

Hypervariable sequence

np

Nucleotide position

mtDNA

Mitochondrial DNA

D-loop

Displacement loop

ROS

Reactive oxygen species

OXPHOS

Oxidative phosphorylation

Notes

Acknowledgments

Authors are grateful for the cooperation received from all patients and healthy individuals. We acknowledge the technical help from Mr. Badal Dey. This work was supported by Indian Statistical Institute.

Conflict of interest

None

Supplementary material

13277_2014_2937_Fig2_ESM.gif (42 kb)
Supplementary Figure 1

RNaseP is a single copy nuclear gene that was used as nuclear DNA standard. ND1 is a rarely deleted region of the mitochondrion that was used to estimate mitochondrial DNA content. XDEL, DNA sequences from the flanks of 4977 bp deletion region, was used for to amplify specifically mtDNA having 4977 bp deletion. Though several other deletions are possible in mtDNA (marked by thin colored lines), XDEL is specific to 4977 bp deletion (marked by thick red line). Black (

) arrow indicates arbitrary positions of forward and reverse primers used for amplification of RNaseP, ND1and XDEL for Taqman assay. (GIF 41 kb)

13277_2014_2937_MOESM1_ESM.tif (3.7 mb)
High Resolution Image (TIFF 3781 kb)
13277_2014_2937_Fig4_ESM.gif (28 kb)
Supplementary Figure 2

∆Ct (XDEL-ND1) values were drawn against ages (yrs) of patients and controls. Blue dots (

), red dots ( ) and green dots ( ) were values for cancer, leukoplakia and control samples, respectively. Regression lines (blue, red and green) were drawn to check the R2 and P values. (GIF 38 kb)

13277_2014_2937_MOESM2_ESM.tif (3.7 mb)
High Resolution Image (TIFF 3777 kb)
13277_2014_2937_Fig8_ESM.gif (0 kb)
Supplementary Figure 3

Number of mutations in D-loop of the cancer patients was drawn against the age (yrs) of them. Regression line shows the value of R2 = 0.0047 and p value = 0.78 (GIF 27 kb)

13277_2014_2937_MOESM3_ESM.tif (2.7 mb)
High Resolution Image (TIFF 2782 kb)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Sayantan Datta
    • 1
    • 3
  • Esita Chattopadhyay
    • 1
  • Jay Gopal Ray
    • 2
  • Mousumi Majumder
    • 1
    • 4
  • Puspita Das Roy
    • 1
    • 5
  • Bidyut Roy
    • 1
  1. 1.Human Genetics UnitIndian Statistical InstituteKolkataIndia
  2. 2.Department of Oral PathologyDr. R Ahmed Dental College and HospitalKolkataIndia
  3. 3.Department of OtolaryngologyJohn Hopkins School of MedicineBaltimoreUSA
  4. 4.Schulich School of Medicine and DentistryUniversity of Western Ontario Medical SchoolLondonCanada
  5. 5.Department of BiochemistryUniversity of CalcuttaKolkataIndia

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